Human herpesvirus-6 (HHV-6) isolation and products

ABSTRACT

A new human B lymphotropic virus, also designated human herpesvirus-6, has been isolated. DNA, molecular clones, antigenic viral proteins and antibodies having specificity to the new virus have been prepared. Various utilities of the new virus and products derived therefrom have been described.

This application is a divisional of and claims the benefit of U.S.application Ser. No. 08/774,118, filed Dec. 23, 1996, which was adivisional of Ser. No. 08/392,674, filed Feb. 22, 1995, now U.S. Pat.No. 5,604,093, which was a continuation of Ser. No. 07/754,220, filedAug. 27, 1991, now abandoned, which was a continuation of Ser. No.07/255,712, filed Oct. 11, 1988, now abandoned, which was a CIP of Ser.No. 07/228,550, filed Aug. 4, 1988, now abandoned, which was a CIP ofSer. No. 06/901,602, filed Aug. 29, 1986, now abandoned, which was a CIPof Ser. No. 06/892,423, filed Aug. 4, 1986, now abandoned, which was aCIP of Ser. No. 06/895,857, filed Aug. 12, 1986, now abandoned, whichwas a CIP of Ser. No. 06/895,463, filed Aug. 11, 1986, now abandoned,the disclosure of which is incorporated by reference.

The present invention is related generally to the isolation andcharacterization of a new virus. More particularly, the presentinvention is related to providing a biologically pure, isolated human Blymphotropic virus, molecular clones, nucleic acid, distinctiveantigenic proteins and a method for detecting antibodies to the newvirus. A virus of the type as described herein has not heretofore beenknown or characterized. The nature, properties, importance and variousutilities of the new virus are now presented.

A virus, designated as human B-lymphotropic virus (HBLV or HHV-6 forhuman herpesvirus-6 ), was isolated from the peripheral bloodlymphocytes of six individuals: one HTLV-III(HIV-1 ) seropositivepatient with AIDS-related syndrome, 1 HTLV-III seropositive patient withangio-immunoblastic lymphadenopathy, 1 patient with dermatopathic, apatient with Mycosis fungoides, a patient with immunoblastic lymphoma,and 1 patient (GS) with acute lymphoblastoid leukemia (Table 1). All sixisolates were closely related by antigenic and molecular analysis, andsera from all 6 virus positive patients reacted immunologically witheach virus isolate (Table 1). In contrast, only 4 sera from more than200 randomly selected healthy donors were seropositive. Subsequent testsshowed a high number of normal blood donors had titers to HHV-6 (59.5%).It was found that HBLV contains a large double-stranded DNA genome, andis morphologically similar to some members of the human herpesvirusgroup. A detailed morphological analysis of HBLV is given below.

It selectively infects freshly isolated human umbilical cord bloodlymphocytes, B-cells and T cells, where it induces the appearance ofcharacteristic large, retractile mononucleated or binucleated cellscontaining nuclear and cytoplasmic inclusions bodies. HBLV isdistinguishable from all known human and sub-human primate herpesvirusesby host range, biological effect on infected cells, and by a lack ofimmunologic, antigenic and genomic relatedness (Tables 2 and 3).

Despite morphological similarities, the host range of HBLV was differentfrom all other members of the human herpesvirus group. For example,initial attempts to transmit the virus to a number of T and Blymphoblastoid cell lines, and to a variety of other cell types, wereunsuccessful, but later tests showed that B- and T-cells, megakaryocytesand neural cells could be infected with HBLV. In contrast, Epstein-Barrvirus (EBV) infects most B cells and some epithelial cells. Furthermore,other herpesviruses [e.g., cytomegalovirus (CMV), Herpes Simplex I andII (HSV) and Varicella-Zoster virus (VZV), infect a variety of celltypes, often inducing cytopathic effects. Immunological comparisons withEBV further emphasized these differences. For example, no EBV nuclearantigens were detected in HBLV-infected cord blood mononuclear cells.

The virus of the present invention has been designated humanB-lymphotropic virus (HBLV) because the virus was intially cultured fromB-cells (the cells had cytoplasmic immunoglobulins), because the virusinitially infects B-cells in vitro in cord blood cultures and becauseHBLV DNA sequences were found in only 3 lymphomas and all 3 were ofB-cell origin. Comparative morphological features which distinguish HBLVfrom other human herpesviruses are listed in Table 4.

For the identification and isolation of HBLV, fresh peripheral bloodmononuclear cells from AIDS patients with associated lymphoproliferativedisorders were established in cell culture (Table 1). In the cultures ofeight patients, primary cell cultures contained a small number of large,refractile mononucleated or binucleated cells which survive for shortperiods of time. These cells frequently contained intranuclear and/orintracytoplasmic inclusion bodies. Electron microscope examinationrevealed that these cells were infected by a DNA virus, 200 nm indiameter (FIG. 3). These large cells were also the only ones in cultureexpressing viral antigens, as measured by fixed and unfixed cellindirect immunofluorescence assays (IFA) (FIG. 2) and by in situhybridization (FIG. 1). All three virus-positive patients werehomosexual males (2 white and 1 black, between the ages of 35 and 44),who were seropositive for HTLV-III with AIDS-pneumocystic pneumonia,with Kaposi's sarcoma, and with undifferentiated B-cell lymphoma.

The presence of the unique large, refractile cells suggested the needfor further examination of patients demonstrating morphologicallysimilar cells in fresh tissues or culture.

HBLV from all six patients could be transmitted to freshly isolatedhuman leukocytes from umbilical cord blood, adult peripheral blood, bonemarrow, and spleen (previously stimulated with PHA-P(phytohemagglutinin-purified)). After in vitro infection the largerefractile cells, noted in primary cultures, appeared within 2-4 dayspost infection. These cells eventually became the predominant cells inthe culture, surviving for an additional 8-12 days. During this time theother cells in the culture rapidly died. As in primary cell cultures,these large cells expressed viral nucleic acids as shown by in situhybridization (FIG. 1), and viral antigens as detected by IFA(immunofluorescent antibodies) (FIG. 2). Virus production was confirmedby electron microscopy (FIG. 3). HBLV-infected cells were typed forsurface markers defined by specific monoclonal antibodies.

Molecular probes which were derived from HSV-1 (cross reactive withHSV-2), CMV, EBV and VZV were used for comparisons with HBLV. While eachindividual viral probe hybridized to its homologous nucleic acids, HBLVwas clearly distinct from these human herpesviruses (FIG. 10).Furthermore, the size of the HBLV genome was shown to contain a minimumcomplexity of 110 kb-pair as determined by analysis of sucrose gradientpurified viral DNA. Finer analysis indicates the genomic size to beabout 170 kb. This genome size, as well as other features (such asmorphology), also distinguished HBLV from DNA viruses of the adenovirus,polyomavirus, papovavirus, and papillomavirus groups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of in situ hybridization of HBLV-infected humancord blood cells using pZVH14 HBLV probe.

FIG. 2A is an immunofluorescent analysis of HBLV-infected acetone fixedcells;

FIG. 2B shows HBLV-infected live cells expressing membrane fluorescenceusing HBLV antibody; and

FIG. 2C shows immunofluorescence of HBLV-infected cells with serumlacking HBLV antibody.

FIG. 3 is an electron micrograph of HBLV showing extracellular envelopedparticles, the insert represents a virus particle showing envelope,spikes, core, capsid and tegument.

FIGS. 4A, B, and C are an electron micrographs of HBLV (negativelystained).

FIGS. 5A and B are Southern blot analyses of HBLV genomic DNA, lanes 1and 2 are positive for HBLV and lane 3 is negative. FIG. 5A: Hind IIIdigested HBLV genomic DNA. FIG. 5B: EcoRI digested HBLV genomic DNA.

FIG. 6 shows HBLV proteins after radioimmunoprecipitation using apositive patient (GS) serum and two dimensional (2D) gelelectrophoresis. HBLV specific proteins are indicated by arrowsaccording to apparent molecular size in KDa.

FIGS. 7A and B show the one dimensional (1D) gel electrophoresispatterns of proteins recognized by human and rabbit anti-HBLV serum byradioimmunoprecipitation. FIG. 7A: 3 hrs ³⁵ S Cysteine labeled HSB-2infected cells. FIG. 7B: Identification of 120 kd protein using HBLVpositive serum.

FIG. 8 shows restriction enzyme map of HBLV clone pZVH14.

FIGS. 9A and B show Western blot analyses of HBLV proteins. FIG. 9A:Concentrated HBLV from HSB 2 cells. FIG. 9B: HSB 2-Cell Lysates.

FIG. 10 shows dot blot analysis of various herpesviruses, showingspecificity for the probes to their genomic DNA.

FIGS. 11A, B and C show Southern blot using pZVH14 probe for detectingHBLV in three human B-cell tumors. FIG. 11A: HBLV sequences in afollicular large cell lymphoma. FIG. 11B: Detection of HBLV sequences inan African Burkitt tumor. FIG. 11C: Detection of HBLV sequences inMulticentric Tumors arising in a Sjogren's Syndrome patient.

FIG. 12 shows restriction enzyme bands generated using Eco R1 and BamH1as visualized on a 0.8% agarose gel using ethidium bromide staining.

FIGS. 13A and B show restriction endonuclease comparison of a HBLVisolate (HBLV Z29) obtained from the Center for Disease Control and theprototype isolate HBLV(GS). Arrows show the restriction enzymedifferences in the EcoR1 restriction patterns between the two isolates.FIG. 13A: Ethidium Bromide Straining. FIG. 13B: Hybridization of HHV6Z29and HHV6 HBLV to HBLV probe pzVH14.

FIG. 14 shows the silver stained gel with enriched HBLV proteins.

FIG. 15 is a Western blot of the gel run in parallel with gel of FIG.14. Clearly 120 and 72 KDa proteins are detected.

FIG. 16 is a map of HBLV clone pZVB70.

FIG. 17 shows HBLV infected human umbilical cord blood lymphocytes.Large refractile infected cells are prominent.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference. Unless mentioned otherwise, thetechniques employed herein are standard methodologies well known to oneof ordinary skill in the art.

The term "substantially pure" as used herein means that the product isas pure as can be obtained by standard methodology conventional in theart.

Despite morphological and other properties similar to some of theherpesviruses, human B lymphotropic virus (HBLV) is a new humanherpesvirus. It is distinguishable from other viruses by biologicalproperties and by the lack of immunological and genomic homology. HBLVis highly lytic in vitro, as are CMV, HSV, HVS (Herpesvirus simia), andHVA (Herpesvirus ateles), but has a different host range than theseviruses. It is possible that HBLV could indirectly cause abnormalitiesin B-cells leading to malignancy in vivo.

Even though in certain instances HBLV was associated with humanT-lymphotropic virus-III/lymphadenopathy-associated virus (HTLV-III/LAV)seropositive donors, other evidence indicates that it is not exclusivelyan AIDS-associated agent. Not only did all HTLV-III seropositivepatients have complicating lymphoproliferative disorders, but HBLV wasalso isolated from a HTLV-III seronegative ALL (acute lymphocyticleukemia) patient. Furthermore, some seroepidemiological analyses haveshown a reactivity clearly dissociated from HTLV-III antibody positiveindividuals.

Serological comparisons demonstrate the uniqueness of HBLV.Immunofluorescence assay was developed following techniques originallydescribed for herpesviruses, and was used to analyze patients andhealthy control sera, and to monitor infected cells. Sera from all sixHBLV positive patients demonstrated an IgG antibody titer to viralcapsid antigens (>1:20). In contrast, only 4 of the more than 200 serafrom randomly selected healthy donors were positive. Subsequentserological surveys indicate the prevalence of HBLV antibodies in normalpopulation to range from about 9% to about 47% with regionaldifferences. The pattern of immunofluorescent staining in fixed,infected cells varied from punctate nuclear staining to diffuse stainingof the entire cell (FIG. 2A). In live cells, the staining was confinedto the cell membrane either as a partial ring or in a capped form (FIGS.2B and C). Uninfected cord blood mononuclear cells were negative whentested with sera from the 6 HBLV positive patients. Sera from thesepositive patients also contained antibody to EBV and CMV. A carefulcomparison of the titers of antibody to EBV, CMV, and HBLV yielded adistinct titer for HBLV as compared to that for EBV and CMV.Furthermore, the reactivity to EBV, CMV, HSV-1 and 2 and VZV wascompletely removed by adsorption with disrupted, EBV-infected cells orwith purified viruses, without significantly affecting the antibodytiter to HBLV.

Sucrose gradient purification of HBLV. Heparinized peripheral bloodleukocytes or human umbilical cord blood mononuclear cells are banded inFicoll-Hypaque and established in cell culture at 36° C. following PHA-P(5ug/ml) stimulation for 48 hours. The cells are then grown in RPMI-1640medium supplemented with 10% fetal bovine serum (heat inactivated, 56°C. for 30 min.) and 5ug/ml hydrocortisone. Frozen supernatants obtainedfrom the infected cells are thawed, collected in 250 ml tubes and spunat 3500 rpm in a Sorvall GSA rotor at 5° C. for 10 min. The clarifiedsupernatants are transferred to SW28 tubes and spun and pelleted at17,000 rpm for 90 min. at 5° C. Pellets obtained are resuspended in 10mM Tris-HCl pH 7.4, 10 mM NaCl, 1 mM EDTA (TNE) to a volume of 300microliters and layered onto a 15-60% sucrose gradient and spun in anSW41 rotor (Beckman) at 20,000 rpm for 30 min. at 5° C. Fractions of 1ml are collected from the top of the gradient. Each fraction is dilutedto 10 ml, spun, and pelleted in an SW41 rotor at 17,000 rpm for 90 min.Pellets are resuspended in 300 microliters of TNE and aliquots assayed(by ELISA and Western Blot) for the presence of virus and for virusinfectivity. Human B Lymphotropic Virus is easily detected in fractions4-9 with a peak in fractions 5-7 by both assays. Extraction of nucleicacids from each fraction shows the presence of double stranded DNA infractions 5-9 with a peak in fraction 7. Virus is also detected byelectron microscopy in the SW41 gradient pellet as well. Virus purifiedfrom fresh unfrozen supernatants according to this procedure is used fordetailed electron microscopy.

Aliquots of the sucrose gradient fractions can be definitively assayedfor the presence of HBLV by DNA dot blot analysis using the pZVH14 9 kbinsert (FIG. 8) as a probe. The pZVH14 molecular clone is obtainablefrom the American Type Culture Collection under Accession No. 40247.

The immunofluorescence, Western blot and radioimmuno-precipitationassays are also employed for detecting HBLV infection and HBLVantibodies in a variety of hematopoietic malignancies, including B-celllymphomas of both AIDS and non-AIDS origin. The presence of HBLVantibodies is elevated in the following disease groups, but theinvention is not intended to be limited to these specific diseases:

Roseola (Exanthum subitum)

Burleitt's lymphoma

Hodgkin's disease

Mononucleosis-like syndromes

Sarcoidosis

Sjogren's Syndrome

A newly described infectious disease syndrome similar to that seen inLake Tahoe characterized as an "acute mononucleosis-like syndrome" inadults, commonly known as chronic fatigue syndrome (CFS).

ALL (acute lymphocytic leukemia) as diagnosed in children of Japanese,Caribbean and African origin.

HIV-1 antibody positive AIDS, ARC and PGL (persistent generalizedlymphadenopathy) patients.

HBLV Virus Propagation. Infection of human umbilical cord blood orperipheral blood mononuclear cells is conducted by cell-freetransmission as follows:

1) Fresh blood samples are diluted 1:1 with RPMI-1640 and spun (andbanded) on a Ficoll gradient.

2) The banded mononuclear cells are washed and put into culture in thepresence of PHA-P (5ug/ml) and hydrocortisone (HC) (5ug/ml) in 20% fetalcalf serum (FCS) and RPMI-1640.

3) After 24 hours, polybrene (2ug/ml) is added to the culture and after6-24 hours, the cells are pelleted.

4) A one ml aliquot of freshly harvested or frozen infected culturesupernatant is added to the pellet and incubated at 37° C. for 1-2 hour,with frequent agitation.

5) Fresh medium [10% FCS and HC (5ug/ml) in RPMI-1640] is then added tothe suspension, cultured, and incubated at 36° C.

6) Within 2-10 days post infection, the characteristic enlargedrefractile cells become visible. Supernatant is harvested at the peak ofinfection as measured by immunofluorescence and by visual observation ofthe culture for further transmission.

Cells infected by HBLV were also used to directly compare immunologicalcross-reactivities with other human and nonhuman primate herpesvirusesusing specific monoclonal antibodies, hyperimmune sera, or sera fromantibody positive control donors. As summarized in Tables 2 and 3,monoclonal antibodies to EBV, CMV, HSV, and hyperimmune sera to RhesusCMV and African Green CMV, did not react with HBLV-infected cells. Humansera possessing antibodies to EBV, CMV, HSV, and VZV also did not reactwith HBLV-infected cells. Furthermore, sera from several old World andNew World primates, many of which had antibodies to nonhuman primateherpesvirus (including EBV-like viruses and CMV), did not show anycross-reactivity with HBLV-infected cells (Table 2).

Immunofluorescent Analysis of HBLV-infected cells. A modification of theindirect immunofluorescence assay developed by Henle et al (J.Bacteriol.91:1248-1256) for EBV was used for the detection of antibodyto HBLV capsid antigens. For this assay, HBLV-infected cord bloodmononuclear cells were isolated by Ficoll gradients to remove deadcells. Uninfected human cord blood mononuclear cells were used ascontrols. Uninfected and infected cells were washed 3 times for 10minutes with PBS without Mg++Ca++, resuspended in PBS containingMg++Ca++, deposited on Teflon coated slides, air dried, and fixed incold acetone for 10 minutes. Patient's sera (heat inactivated at 56° C.for 30 minutes and clarified by centrifugation) were added to theacetone fixed cells, incubated in a humidity chamber at 37° C. for 40minutes, washed with PBS, air dried, and stained with affinity purifiedFITC conjugated anti-human IgG (H and L) for 40 minutes. The cells werecounterstained with Evans blue (1:500 dilution in PBS) for 5 min tofurther reduce background due to autofluorescence. The cells were againwashed as above, air dried, and mounted with IFA (immunoflourecenceassay) mounting solution. Large cells with greenish to yellow granularimmunofluorescent and cytoplasmic staining were scored as positive cellsfor HBLV. The example of assays carried out 5 days post infection areshown in FIG. 2a. Small cells in the background did not react withpatient serum (FIG. 2b with arrows).

As is shown in FIG. 2, detection of viral membrane antigen HBLV infectedas well as uninfected live cells (non-fixed) were washed 3 times inserum-free RPMI1640 medium and treated with patient's serum for 30minutes at 4° C. The cells were again washed, treated with affinitypurified FITC anti-human IgG for another 30 minutes, washed in mediumagain and examined for membrane fluorescence. HBLV infected cells showedsurface markers when tested with patient serum using theimmunofluorescence technique (FIG. 2b).

Southern blot analysis of HBLV genomic DNA. Supernatant fluid from HBLVinfected umbilical cord blood cells was layered onto 20% glycerolcushions and pelleted by centrifuging at 25,000 rpm for 3 hr. in aBeckman SW41 rotor at 4° C. The pellets were suspended in TNE buffer (10mM, Tris-HCl, pH 9; 100 mM, NaCl; 1 mM EDTA), and extracted with PCI9(Phenol:Chloroform:Isoamyl alcohol; 50 mM Tris-HCl, pH9; 100:100:1:10 ::v:v:v:v) followed by Chloroform:isoamyl alcohol (24:1::v:v).Substantially enriched viral DNA was precipitated by adding 2 volumes of95% ethanol. DNA was digested with Hind III and cloned into theBluescribe vector (commercially available from Vector Cloning Systems,Calif.). Several clones obtained were prepared as radiolabeled probesand screened for specificity of hybridization by Southern blotting toHBLV infected human umbilical cord blood cell DNA and by in situhybridization to such infected cells. Results of hybridization of HBLVclone pZHV14 to DNA from pelleted virus digested with Hind III and EcoRlare shown in FIG. 5. Extracellular virus is shown in lane 1, virusinfected human umbilical cord blood cells in lane 2 and negative controlDNA isolated from the skin of an AIDS patient in lane 3. Clone pZVH14scored positive in these assays and did not hybridize to uninfectedcontrols. The infected cell DNA shown in lane 2 is isolated insubstantially pure form after several rounds of cell free virustransmission in human umbilical cord blood cells.

In addition to the procedures described above, the following specificmethods and materials may also be employed.

Rather than using cord blood cells, HBLV can also be propagated byinfecting other suitable host cells such as HSB2 cells obtainable fromATCC (CCL 120.1). HBLV(GS) strain was collected from 15 liter culturesof infected HSB2 cells by continuous flow centrifugation onto 10% to 60%sucrose gradients. Bands collected between 1.135 and 1.210 g/ml werepelleted at 20,000 rpm and resuspended in PBS containing 1 mMphenylmethylsulfonyl (PMSF) and 10 mM MgCl₂. The suspended virions weresubjected to six strokes in a Dounce homogenizer and 23 units per ml ofRNAse free DNAse (Boehringer-Mannheim) and incubated for 10 min. at 37°C. The total volume (2ml) was layered onto 36 ml 5-30% dextran T10gradient (w/w) in 0.5 mM phosphate buffer, pH7, and centrifuged in aBeckman SW 27 rotor for 1 hr. at 20,000 rpm at 4° C. (Dolyniuk et al, J.Virol. 17:935, 1976). Fractions of 4 ml were collected and a visibleband was collected in fractions 7-9. Examination of fraction 10 underthe electron microscope revealed highly enriched virions with verylittle cellular debris. Electron microscope examination of virionsfiltered through 0.2% polyvinylpyrollidone (PVP) treated 0.45 um Nalgenefilters also gave excellent results and protein gel analyses showed apurification indistinguishable from fractions 7-9 above by electronmicroscopy (FIG. 3).

Purification of HBLV genomic DNA

Infection and banding of the virions by continuous flow centrifugationwas as described herein supra. The sucrose-banded virus was pelleted at20,000 rpm in a Beckman SW27 rotor for 90 min. The virus was resuspendedin 400 ul of TE buffer (20 mM, Tris-HCl, 1 mM EDTA) and 130 ul of 10%sodium lauryl sarkosinate added. The viral lysate was incubated at 60°C. for 20 min. and then layered onto a 54% CsCl, 0.1 mg/ml ethidiumbromide solution and centrifuged in a Beckman SW50 at 45,000 rpm for 20hr. at 20° C. The viral DNA band (1/3 from the top of the gradient) wasvisualized under UV illumination and removed by side puncture with aneedle and syringe. The HBLV DNA-CsCl aliquot was extracted 5 times withequal volume of n-butanol and then dialyzed against 2 changes of 1000 mlof TE buffer at 4° C. Dialysis membrane was placed over an Eppendorftube and held in place with an Eppendorf cap into which a hole had beenbored. The tube was inverted and floated on the buffer for dialysis. DNAprepared in this way was substantially pure to visualize theethidium-stained restriction digests on agarose gels and for thecreation of plasmid vector libraries. The DNA yield is usually greaterthan 30 ug per 25 liters of cell free supernatant depending on theextent of the infection.

Labeling of cells

Media for 24 hr. labeling incubations was prepared by mixing 8 ml ofmethionine free DMEM (D-Met) (Gibco), 2 ml of 50% fetal calf serum inRPMI 1640 and 0.1 ml gentamicin (100×concentrated, 5 mg/ml). Media for2-3 hr. labeling incubations contained D-Met and 10% fetal calf serum.The amount of 5 mCi of [³⁵ S]methionine (or other radiolabeled aminoacid) was lyophilized and reconstituted with 400 ul of D-Met. Cells inthe amount of 5×106 were pelleted at 1000 rpm for 5 min. in the SorvallGLC bench top centrifuge and resuspended in labeling media. For 24 hr.labeling, the cells were split into two 0.8 ml aliquots in a 24 wellmicrotiter plate and 50 ul of the reconstituted [³⁵ S]methionine wasadded to each. For 2-3 hour labeling, 5×10⁶ cells were resuspended in1.0 ml of labeling medium and split into two 0.5 ml aliquots and 50 ulof radiolabeled methionine added to each. Cells were incubated at 37° C.under 5% CO₂ and 85% humidity for the period of time necessary forlabeling.

Radioimmunoprecipitation

After metabolic labeling, (as described herein supra) the cells werediluted in 10 ml of ice-cold phosphate buffered saline (PBS) andpelleted for 5 min. at 1000 rpm in the Sorvall GLC bench top centrifuge,resuspended in 10 ml of fresh ice-cold PBS and pelleted a second time.The cells were resuspended in 1 ml of PBS and transferred to anEppendorf tube and centrifuged at half maximal speed for 2 min. About550 ul of lysis buffer [0.1% SDS (sodium dodecyl sulfate), 1% TRITON,X-100, (T-octylphenoxypolyethoxyethanol, Sigma Chemical Company, St.Louis, Mo.) 1% desoxycholate (free acid), 20 mM Tris-HCl, pH 8.0, 150 mMNaCl and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma)] was added.The lysate was vortexed at a setting of 5 for 15 sec., allowed to sit onice for 10 min. and vortexed again. The samples were then centrifuged attop speed in an Eppendorf centrifuge for 3 min. A 50 ul stock aliquotwas removed from each tube and immediately frozen on dry ice. Theremaining supernatant was transferred to a clean Eppendorf tube and 20ul of sera was added. The tubes were placed on a rotor at 4° C. andgently inverted for 12 hr. The samples were then centrifuged at topspeed for 2 min. and all but 10 ul of the supernatant was removed to anew Eppendorf tube. The amount of 100 ul of a 50% (v/v) slurry ofprotein A SEPHAROSE, beaded agarose (Pharmacia) in lysis buffer wasadded to each tube and the tubes gently inverted for 30 min. The sampleswere centrifuged for 2 min. at top speed and the supernatants discarded.The protein A pellet was washed 6 times by resuspension in lysis bufferand centrifuged for 15 sec. at top speed. After removal of thesupernatant of the sixth wash, the pellet was frozen and sent to ProteinData Bases, Inc. (a commercial analytical service laboratory inHuntington Station, N.Y.) for the gel runs.

All radioimmunoprecipitations were performed using serum from patientGS, the source of the prototype HHV-6 isolate. Specificity of theantisera was demonstrated by adsorbing the sera against virionpreparations of human cytomegalovirus, Epstein-Barr virus, VaricellaZoster virus, and Herpes Simplex type 1.

High resolution 2 dimensional gels (HR2D) of HHV-6 Proteins

The viral proteins were prepared by SDS-BME (sodium dedecylsulfate-basic maintenance emulsion) lysis of gradientbanded virions andRNA-DNAse treatment as described herein supra and then frozen on dry iceaccording to the standard protocols of Protein Data Bases Incorporated(PDI), Huntington Station, N.Y. The samples were run at PDI on 12.5%broad range non-equilibrium and equilibrium polyacrylamide gels andsilverstained. The protein-A Sepharose bound radiolabeledimmunoprecipitates run on 12.5% broad range non-equilibriumpolyacrylamide gels were then exposed for autoradiography at PDI.

It should be noted that in addition to radioimmunoprecipitation (RIP),Western blot, indirect immunofluorescence assay (IFA) enzyme linkedimmunosorbent assay (ELISA), and the like can also be utilized to detectviral antigens or antibodies. These techniques are well established andknown to one of ordinary skill in the art to which this inventionbelongs.

HR2D Western blotting

Immunoblotting was performed after HR2D electrophoretic resolution offractions of HBLV prepared from sucrose gradients or filtered virus asdescribed herein supra. The nitrocellulose sheets were stored at 20° C.prior to use. Sheets were incubated for one hour in a blot solution of4% normal goat serum, 4% fetal bovine serum, 5% non-fat dry milk and0.02% thimerosal for blocking. Sheets were then incubated with serumfrom a known HBLV infected patient, diluted 1:1000 in the blot solution.After 3 successive 5 minute washes with PBS, they were reacted insequence for 1 hour with 1:500 dilution of affinity purified goatanti-human IgG labeled with biotin and for one hour with 1:1000 ofhorseradish peroxidase streptavidin (Kirkegaard and Perry Labs., Inc.,Gaithersburg, Md.) at room temperature (about 21°-25 C.) in 5% normalgoat serum in PBS and 0.02% Thimersol. A stock solution of4-chloronaphthol (4CN stock) was prepared by dissolving 0.3 g of4-chloronaphthol in 100 ml of methanol. Staining was carried out in asolution containing 2 ml of 4CN stock, 8 ml of PBS and 4 microliters ofhydrogen peroxide. The reaction was stopped by washing with distilledwater.

In order to obtain a better size estimate, DNA was purified from viruscollected by continuous flow centrifugation from 15 liters of HBLVinfected HSB2 cell culture supernatant and pooled. The regions of the10% to 60% sucrose gradient pooled were from 1.14 to 1.17 g/ml, fractionA, and from 1.17 to 1.21, fraction B. The virions were pelleted, lysedand the DNA purified by banding on cesium chloride gradients anddialyzed. FIG. 12 shows the restriction enzyme bands generated usingEcoRI and BamHI as visualized on 0.8% agarose gels by ethidium bromidestaining. Over 26 bands were generated by EcoRI digestion (A to Z, topto bottom) and at least 15 fragments with BamHI (A to O). The bands seenwere of similar intensity with a marked absence of submolar fragments.compared to other herpesviruses. Possible exceptions were the EcoRI A'and the BamHI F' and M' fragments which had intensities equivalent to1/4 M. The reasons for the generation of these bands are not understood;however, they are possibly due to genomic inversions and were notcounted for the genome size estimates. Table 5 shows the results ofrestriction enzyme analyses of HBLV.

The construction of BamHI plasmid libraries from the DNA showed thatnearly 100% of the fragments cloned were HBLV thereby providing furtherevidence that the bands visualized in FIG. 12 can be used as a reliableestimate of the HBLV genome size. The molecular weights of the fragmentslisted in Table 5 gave genome size estimates of 168,000 and 172,000 forthe EcoRI and BamHI digests, respectively. By this estimate, the genomeof HBLV is approximately the size of the Epstein-Barr virus genome.

Restriction endonuclease comparison of another independent HBLV isolate,HBLV(Z29), to the prototype HBLV(GS) strain is shown in FIG. 13. Thearrows indicate the areas where the EcoRI digests of each stain differas visualized by ethidium bromide staining. Hybridization to one HBLVprobe, ZVH14, revealed identical restriction patterns between the twoisolates; however, by probe ZVB70, the HBLV(GS) BamHI B fragment, showeddifferences (not shown). This indicates that restriction siteheterogeneity can be observed among different isolates of HHV-6 .Another isolate, HBLV(DV), was identical by hybridization with both theZVH14 and ZVB70 probes to HBLV (GS).

Studies of the complexity of the enveloped HHV-6 proteins were attemptedby banding the virus collected by continuous flow centrifugation onDEXTRAN T-10 gradients similar to methods used to purify the proteins ofthe enveloped EBV (Dolyniuk et al, 1976, supra). The virions obtainedfrom continuous flow centrifugation were pelleted, treated with DNAse 1and then banded on 10% to 30% DEXTRAN T-10 gradients. The variousfractions collected from the top were analyzed by electron microscopyand the virus was pelleted from those which looked relatively free ofcellular debris. A viral band was seen toward the bottom of the gradient(fractions 7-9) and the fraction immediately below (fraction 10) wasconsidered to be relatively free of cellular debris when compared tovirus obtained after a single banding. The virus was found in clusterswith little cellular material. Virus prepared by this method wheninoculated into rabbits resulted in the generation of HBLV specificantibodies in 14 days which were readily detected by indirectimmunofluorescence assay on infected cells. Subsequent bleeds gave somenon-specific cellular background in IFA tests. Hence, the animals shouldbe bled about 14 days post inoculum. These antibodies can be utilizedfor detection of HBLV by established techniques.

The pure preparations of the virus as revealed by electron micrographs,were used to determine the proteins by direct visualization on highresolution two dimensional polyacrylamide gels (HR2D). As mentionedherein supra by procedures developed at Protein Data Bases, Inc.,several 12.5% broad range non-equilibrium gels were run and a gel of thevirions obtained from the DEXTRAN T-10 fraction 10 was silverstained(FIG. 14). A parallel gel was run and Western blotted using GS serum andperoxidase conjugated goat anti-human antibody. Two major proteins weredetected at 120 and 72 kDa as shown in FIG. 15. Radioimmunoprecipitationof HBLV-infected cell lysates with the GS serum showed severaladditional proteins (FIG. 6). In addition to the proteins detected byWestern blots, radioimmunoprecipitations performed on proteins fromlysates of metabolically labeled cells showed a protein at 120 kDa;however, additional major proteins at 200, 80 and 19 kDa, as well assome minor proteins at 60 kDa, 80 kDa and several in the 30 kDa rangewere also detected. Two forms of the 19 kDa proteins were observed, amore acidic form, 19a, and a more basic major form, 19b, possibly due todifferences in phosphorylation (FIG. 6). The antigenic proteins can thenbe isolated in substantially pure form following standard purificationtechniques, such as column chromatography, HPLC, preparative gelelectrophoresis, and the like. These proteins can be identified, forexample by Western blot using HBLV antibody positive sera. Apharmaceutical composition in accordance with the present inventioncomprises an immunogenic amount of the antigenic protein in apharmaceutically acceptable carrier. Antigenic proteins or portionsthereof can also be obtained from gt11 expression libraries or the like.

Antigenic proteins of the present invention also allow detection of thepresence of HBLV antibodies in a biological sample by reacting saidsample with the viral antigens, a positive antigen-antibody complexformation being indicative of HBLV infection. Antigen-antibody reactionscan be detected by any standard immunological techniques well known toone of ordinary skill in the art, such as radioimmunoassay, Westernblot, ELISA, immunofluorescence, histoimmunological tests and the like.

EXAMPLES

Example 1. Fresh tissue sections from 3 patients were found to contain alow number of HBLV-infected cells. One patient, a 40 year old Hispanicwith a history of IV drug use, was seropositive for both HTLV-I andHTLV-III, and was diagnosed with AIDS-pneumocystic pneumonia withassociated dermatopathic lymphadenopathy. Another was a 61 year oldwhite male who received multiple blood transfusions in conjunction withopen heart surgery 4 years prior to death. This patient was seropositivefor HTLV-III and was diagnosed with immunoblastic lymphadenopathy withsome skin involvement. A third patient (GS) was a 16 year old black malediagnosed with acute lymphocytic leukemia of the T-cell type. Unlike theothers, this patient was seronegative for HTLV-III. Primary peripheralblood mononuclear cell cultures from these patients also contained asmall number of the unique cells which, upon close examination, werealso found to be infected by HBLV.

Example 2. A direct comparison of molecularly cloned sequences of theHBLV genome with the genomes of other herpesviruses was also conducted.Several DNA clones obtained from nucleic acids extracted from purifiedvirus were examined for specificity and for comparison with other DNAviruses. Two HBLV clones, designated pZVH14 (FIG. 8) and pZVB70 (FIG. 16ATCC No. 40473), were used in these studies. Southern blot analysis(FIG. 5) showed the presence of viral specific DNA in Hind-III and EcoRIdigests of DNA from both purified virus and HBLV-infected human cordblood cells. In situ hybridization experiments with the pZVH14 probealso confirmed that these sequences were confined to the infected cells(FIG. 1).

Example 3. Monoclonal antibodies and hyperimmune sera prepared againsthuman and simian herpesviruses were tested for reactivity with HBLVinfected cells by indirect immunofluorescence procedures as describedherein above. Monoclonal antibodies to EBV and HCMV were used at 1:40dilution; HSV-I and II, VZV and HVS at a 1:10 dilution and normalascites fluid was used at 1:5 and 1:10 dilutions. Hyperimmune sera toAfrican green and Rhesus monkey CMV were heat inactivated (50° C. 30min.), clarified at 10,000 rpm, and then were used at 1:10 dilutions. Inaddition to the sera shown, human sera containing antibodies to EBV,CMV, HSV-I and II, and VZV also did not react with HBLV infected cells.African green monkey and Rhesus sera containing antibody to CMV werealso negative when tested with HBLV. Monoclonal antibodies to EBV andHCMV, and ascites fluid from normal mouse were gifts from Dr. GaryPearson, School of Medicine, Georgetown University, Washington, D.C.Monoclonal antibodies to VZV and HVS were obtained from Dr. Nancy Chang,Baylor College of Medicine, Houston, Tex., and Dr. John Dahlberg, NCI,Bethesda, Md., respectively. HSV-I and II monoclonal antibodies werepurchased from Dupont, Boston, Mass. Hyperimmune serum to purifiedAfrican green and Rhesus CMV were previously prepared in rabbits by Dr.Ablashi. The specificity of the serum containing antibodies to HBLV wasshown by adsorbing it against the other human herpesviruses (eitherwhole virus or infected cells).

Abbreviations used: HBLV, Human B lymphotropic virus; EBV, Epstein-Barrvirus; HCMV, Human cytomegalovirus; HSV, Herpes simplex virus; VZV,Varicella-Zoster virus: HVS, Herpes virus saimiri, VCA (Viral capsidantigen); EA, early antigen; MA, membrane antigen.

HBLV infected cord blood mononuclear cells were stained with an HBLVnegative serum resulting in a considerable number of large cells with noimmunofluorescence.

Example 4. Serum from Old World and New World primates were tested forantibody to HBLV by indirect immunofluorescence as described.

Some sera from the Old World primates were gifts from Dr. P. Kanki,Harvard School of Public Health, Boston, Mass. All sera were heatinactivated at 50° C. for 30 minutes, and clarified by centrifugationbefore use. HBLV-infected cord blood leukocytes, P3HR-1 (an establishedcell line expressing EBV-VCA), and Owl monkey kidney cells infected byHSV-strain II were used for comparisons. When infected cells showedcytopathic effects, the cells were fixed in acetone and used for the IFAtest.

Three owl monkeys and one cottontop marmoset were previously inoculatedwith HVS. Sera from these Animals possessed antibody to HVS late antigenwhich cross-reacted with Herpesvirus ateles. The results are presentedin Table 2.

Example 5. In situ hybridization of HBLV-infected human cord bloodcells. Tests were performed utilizing ³⁵ S-labeled RNA probes asdescribed herein supra. Clone pZVH14 of the HBLV genome was used as atemplate for radiolabeled RNA using T7 RNA polymerase, ³⁵ S-labeled GTP,and unlabeled ribotriphosphates. Less than one grain per cell wasobserved in uninfected negative control cultures. Large refractile cellscharacteristic of the infected cultures were heavily labeled, indicatingthe expression of abundant viral messages (FIG. 1).

Example 6. Two dimensional gel electrophoresis patterns of proteinsrecognized by human sera against human B cell lymphotropic virus (HBLV)are shown in FIG. 6. Human umbilical cord blood lymphocytes or HSB₂cells were infected with HBLV and then labeled by incubation with ³⁵S-methionine for periods of either 3 hours or 24 hours. H9 cells wereused as negative controls. The labeled cells were lysed and the proteinsimmunoprecipitated according to established procedures (Protein DataBases, Inc., New York). Spotsseen on the gels of the lysates frominfected cells but not seen on the control gels represent candidatevirus proteins arrayed in unique virus specific patterns. These patternsserve as a fingerprint which can specifically identify HBLV. Theproteins detected are antigenic proteins, the coding sequence of whichcan be cloned and expressed, and the purified proteins thus obtained canbe used as diagnostic reagents.

PREPARATION OF THE CLONES

Of course, the availability of the biologically pure HBLV and its DNA,allows the preparation of the clones of HBLV. A general method ofcloning the Human B Lymphotropic Virus (HBLV) genome involves isolatingviral DNA after infection of suitable host cells (such as HSB₂ and thelike), primary cells or cord blood cells with the HBLV virus. Theunintegrated viral DNA is then cloned in a suitable cloning vector suchas a plasmid or a lambda phage to create libraries which can be screenedfor the presence of viral specific DNA fragments.

Infected cells and cultured peripheral cord blood cells produce HBLVvirus and serve as the principal source of the virus for immunologicalassays and the like for detecting virus-specific antigens and antibodiesin human sera. Cultures of infected cells are grown and the virusharvested from the supernatant and the high molecular weight DNAextracted from the virus. This produces viral DNA containing the HBLVgenome of the present invention. This DNA is then subcloned in asuitable plasmid to produce a clone. A complete description of theprocedures for preparing clones can be found in such standardpublications as Maniatis et al: "Molecular Cloning," Cold Spring Harbor,N.Y.

Two elements of the above process are well known are a part of therecombinant DNA procedures: the DNA library and the differentialscreening of DNA inserts to infected and uninfected cells. The libraryis formed by taking the total DNA from the enriched or purified virusDNA, cutting the DNA into fragments with suitable restriction enzyme(s),joining the fragments to plasmid vectors, and then introducing therecombinant DNA into a suitable host. The viral specific DNA fragmentsare distinguished by their hybridization to infected cell DNA and/or byin situ hybridization to infected cells but not to uninfected cells.

As shown herein infra, a molecular clone, pZVH14, of the HBLV genome isuseful as a template for radiolabeled RNA using T7 RNA polymerase, ³⁵S-labeled GTP, and unlabeled ribotriphosphates.

In the preferred embodiment of the present invention, supernatant fluidfrom HBLV infected cells is layered onto 20% glycerol cushions andpelleted by centrifuging at 25,000 rpm for 3 hr. in a Beckman SW41 rotorat 4° C. The pellets are suspended in TNE buffer (10 mM, Tris-HCl, pH 9;100 mM, NaCl; 1 mM EDTA), and extracted with PCI9(Phenol:Chloroform:Isoamyl alcohol; 50 mM Tris-HCl, pH9; 100:100:1:10v:v:v:v) followed by chloroform;isoamyl alcohol (24:1::v:v). Enrichedviral DNA is precipitated by adding 2 volumes of 95% ethanol. DNA isdigested with Hind-III and cloned into the Bluescribe vector(commercially available from Vector Cloning Systems, Calif.). Severalclones obtained after screening with labeled, enriched, DNA wereexamined for specificity of hybridization to the HBLV DNA and by in situhybridization to HBLV-infected cells.

Clones pZVH14 and pZVB70 which were thus produced, scored positive whentested by hybridization techniques and did not hybridize to uninfectedcontrols. The infected cell DNA is isolated after several rounds of cellfree virus transmission in human umbilical cord blood cells or HSB₂cells. Clone pZVB70 was obtained from CsCl gradient banded DNA ofsucrose banded virus. DNA was BamH1 digested as described herein supra.

It is noted that these probes, either alone or in combination, can beemployed for detecting the viral DNA or RNA and virus-infected cellscontaining HBLV nucleic acids by any of several standard techniques wellknown to one of ordinary skill in the art. Examples of such wellestablished techniques are Southern and dotblot for DNA analysis,Northern blot for RNA analysis and in situ hybridization. Furthermore, aprobe for in situ hybridization can be made by any of well establishedprocedures such as radiolabeling or covalent linkage of hapten or enzymeto DNA. A few illustrative examples are now provided.

Example 7. Several DNA clones obtained from nucleic acids extracted frompurified virus obtained as described above, were examined forspecificity relative to other DNA viruses. HBLV clone designated pZVH14,contained a 9.0 kb Hind III fragment. Southern blot analysis showed thepresence of viral specific DNA in Hind-III and EcoRI digests of DNA fromboth purified virus and HBLV-infected human cord blood cells. In situhybridization tests with the same probe also confirmed that thesesequences were confined to infected cells.

Example 8. Human B Lymphotropic Virus clone pZVH14 has been restrictionenzyme mapped as shown in FIG. 8.

Example 9. Similarly, HBLV clone pZVB70 has been restriction enzymemapped as shown in FIG. 16.

It is noted that based on the sequence information, any number ofspecific clones can be generated and used as probes. The techniques arewell established and known to one of ordinary skill in the art to whichthis invention belongs.

Example 10. In situ hybridization of HBLV-infected cells. Tests wereconducted utilizing ³⁵ S-labeled RNA probes as described herein supra.Clone pZVH14 of the HBLV genome were used as a template for radiolabeledRNA using T7 RNA polymerase, ³⁵ S-labeled GTP, and unlabeledribotriphosphates. Less than one grain per cell was observed inuninfected negative control cultures. Large refractile cellscharacteristic of the infected cultures were heavily labeled, indicatingthe expression of abundant viral messages (FIG. 1).

Example 11. Based on the nucleotide sequence, polymerase chain reactiontechnique (Saiki et al, 1985, BioTechnology, 3:1008; Science, 230:1350)was employed to obtain increased levels of nucleic acids from specimens(tissue or cell culture) suspected of HBLV infection from diseased andnormal A (control) populations and the presence of HBLV detected bySouthern blotting of the amplified HBLV DNA or other method of detectingthe amplified DNA with radiolabeled or nonradiolabeled probes as arewell known to one of ordinary skill in the art.

A deposit of the clones pZVH14 and pZVB70 have been made at the ATCC,10801 University Boulevard, Manassas, Va. 20110-2209 under the accessionnumbers 40,247 and 40,473, respectively. The deposit shall be viablymaintained, replacing if it becomes non-viable, for a period of 30 yearsfrom the date of the deposit, or for 5 years from the last date ofrequest for a sample of the deposit, whichever is longer, and madeavailable to the public without restriction in accordance with theprovisions of the law. The Commissioner of Patents and Trademarks, uponrequest, shall have access to the deposit.

In summary, as demonstrated herein, high level production of HBLV cannow be obtained by the use of the HSB2 or other cell lines. This allowspurification of the enveloped virus and the viral nucleic acids.Purification of the viral DNA has been demonstrated by hybridizationwith specific cloned viral DNA such as clone pZVH14. Although the sizeestimate (170,000) of the HBLV genome is similar to that of EBV,evidence by molecular hybridization shows distant relationships to thehuman cytomegalovirus and to the Marek's disease cirrus of chickens(data not shown).

Comparison of the Western blots from the HR2D gels to theradio-immunoprecipitation revealed a major antigenic protein of 120 kDaand other antigenic proteins described herein supra which is detectableby both (RIP and Western blot) methods. The 120 Kd protein seems to be amajor antigenic protein as demonstrated by anti-HBLV patient sera.Increased resolution of the minor proteins on 2D gels indicates that itwould be easier to verify the presence of characteristic viral proteinsby this method than by 1D gels. Very clean background seen in the twodimensional Western blot, in which 120 Kd and 72 Kd proteins weredetected, may be the method of choice.

Although not necessary, because biologically pure virus can be obtainedby following the standard procedures described herein by anyone ofordinary skill in the art, nevertheless a deposit of the isolated virushas been made at the ATCC, Rockville, Md. under accession number VR2225.A deposit of the anti-HBLV positive serum has also been made at the ATCCunder accession number 40476. The deposits shall be viably maintained,replacing if it becomes non-viable, for a period of 30 years from thedate of the deposit, or for 5 years from the last date of request for asample of the deposit, whichever is longer, and made available to thepublic without restriction in accordance with the provisions of the law.The Commissioner of Patents and Trademarks, upon request, shall haveaccess to the deposit.

A diagnostic kit in accordance with the present invention comprisescontainers separately containing anti-HBLV antibodies, one or morepurified or cell associated antigenic viral protein(s) produced by HBLVin any part of its replicative cycle (i.e., HBLV infected cells or cellsexpressing specific HBLV proteins); HBLV specific nucleic acid probes;positive and negative controls and instructional material to performdiagnostic test employing said antibodies, antigenic viral protein(s),probes and the like. Of course, the present invention also allows thedetection of HBLV present in any biological sample. Any suitable methodmentioned herein can be utilized as deemed most appropriate by one ofordinary skill in the art, depending on such factors as the location,nature, amount of the sample available and the like.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

                                      TABLE 1                                     __________________________________________________________________________    ISOLATION OF HBLV FROM PERIPHERAL BLOOD LYMPHOCYTES OF                         PATIENTS WITH LYMPHOMA AND LYMPHADENOPATHY                                                          Serology*                                              Patient                                                                           Description        HTLV                                                                              HBLV                                                                              HBLV Isolation**                               __________________________________________________________________________    1   RC 29 WM                                                                            AIDS         +III                                                                              1:80                                                                              +                                                  KS                                                                            B cell lymmphoma                                                            2 WR 57 WM OHS -- 1:40 +                                                        AILD                                                                        3 PD 40 WM Dermatopathic +II                                                    lymphademopathy; IVDA and 1:80 +                                              T8.sup.+  skin infiltrate +III                                              4 GS 17 BM T-cell ALL (T-4.sup.+) -- 1:160 +                                  5 RW 66 RM Mycoses Fungoides (T-4.sup.+) -- 1:80 +                              Cutaneous T-cell Lymphoma                                                   6 BD 35 BF Immunoblastic Lymphoma -- 1:80 +                                 __________________________________________________________________________     *Serology was done by indirect immunofluorescence using as standard a         reference virus isolated from patient GS.                                     **PBL from patients were cultured as the primary source of virus. Virus       particles were transmitted to fresh human cord blood. Positive cultures       were identified by morphology, IF, and EM.                               

                                      TABLE 2                                     __________________________________________________________________________    Cross-Reactivity of Nonhuman Primate Sera                                                 Virus Used to Infected Target Cells                                           HBLV      EBV       HSV                                             No. Positive No. Positive No. Positive                                        No. Tested (VCA)/No. Tested No. Tested                                        Serum Sources (Percent Positive) (Percent Positive) (Percent Positive)      __________________________________________________________________________    Old World Primates                                                              Chimpanzee 0/5 (0) 5/5 (100%) 0/4 (0)                                         Gorilla 0/3 (0) 2/3 (66.6%) 0/3 (0)                                           Orangutan 0/2 (0) 1/2 (50%) 0/2 (0)                                           Baboons 0/3 (0) 3/3 (100%) 0/3 (0)                                            Stumptail 0/2 (0) 1/2 (50%) 0/2 (0)                                           Rhesus 0/9 (0) 6/9 (66/6%) 0/7 (0)                                            African Green 0/10 (0) 6/10 (60%) 0/10 (0)                                    New World Primates                                                            Squirrel monkeys 0/10 (0) 0/10 (0%) 8/10 (80)                                 Owl monkeys 0/6 (0) 0/6 (0%) 3/6 (50)                                         Marmosets (common) 0/6 (0) 0/6 (0) 0/6 (0)                                    Marmoset (cottontop) 0/3 (0) 0/3 (0) 1/3 (33.3)                             __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Immunological Cross Reactivites of HBLV to Other Human and Nonhuman            Primates Herpesviruses Antibody Viruses Used to Infect Target Cells                                  HSV-I     Af. Gr.                                                                           Rhesus                                    Source HBLV EbV PCMV and II VZV HVS CMV CMV                                 __________________________________________________________________________    ERV Monoclonal Antibody                                                                    -   +  -   -   -  -  -   -                                         (VCA, EA, MA)                                                                 HCMV Monoclonal Antibody - - + - - - - -                                      HSV I and II Monoclonal - - - + - - - -                                       Antibody (early and                                                           late antigens)                                                                VZV Monoclonal Antibody - - - - + - - -                                       (late antigens)                                                               HVS Monoclonal Antibody - - - - - + - -                                       (late anitgens)                                                               Af. Green Monkey - - - - - - + -                                              CMV (hyperimmune serum)                                                       Rhesus Monkey - - - - - - - +                                                 CMV (hyperimmune serum)                                                     __________________________________________________________________________

                  TABLE 4                                                         ______________________________________                                        Morphologic comparison of HBLV with other herpes viruses                        Feature    HBLV      HSV*    HCMV(6) EBV(7)                                 ______________________________________                                        Diameter of                                                                            60-80 nm  50-70 nm  64.3 nm 48 nm                                      nucleoid                                                                      Diameter of 95-105 nm 95-110 nm 106.4 nm 80 nm                                capsid                                                                        Symmetry of Icosahedral Icosahedral Icosahedral Icosahedral                   capsid                                                                        No. of 162 162 162 162                                                        capsomeres in                                                                 capsid                                                                        Thickness of Dense, Often Dense, Variable,                                    tegument prominent, indistinct, prominent, 20 nm                               25-40 nm 20-40 nm 24.4 nm                                                    Diameter of 160-200 nm 150-200 nm 174 nm 120 nm                               enveloped                                                                     virion                                                                      ______________________________________                                         *HSV used for this comparison was prepared simultaneously and under           identical conditions as HBLV.                                            

                  TABLE 5                                                         ______________________________________                                               Fragment                                                                             MW (kb)                                                         ______________________________________                                        EcoR1                                                                                  A        20.0                                                          B 17.0                                                                        C 16.0                                                                        D 10.5                                                                        E 8.0                                                                         F 7.7                                                                         G 7.4                                                                         H 6.6                                                                         I 6.3                                                                         J1,J2 5.9                                                                     K 5.4                                                                         L 5.0                                                                         M 4.5                                                                         N 4.36                                                                        O 3.85                                                                        P 3.75                                                                        Q 3.5                                                                         R 3.25                                                                        S 3.05                                                                        T 2.95                                                                        U 2.5                                                                         V 2.4                                                                         W 2.3                                                                         X 2.25                                                                        Y 2.1                                                                         Z 1.75                                                                         Z1 1.5                                                                        Z2 1.49                                                                       Z3 1.35                                                                       168.55                                                                     BamH1                                                                                  A        40.0                                                          B 30.0                                                                        C 23.1                                                                        D 13.5                                                                        E 11.8                                                                        F 10.9                                                                        G 8.5                                                                         H 6.5                                                                         I 6.2                                                                         J 5.95                                                                        K 5.6                                                                         L 3.4                                                                         M 2.6                                                                         N 2.05                                                                        O 1.95                                                                         172.05                                                                     ______________________________________                                    

What is claimed is:
 1. An isolated polypeptide encoded by a firstnucleic acid which hybridizes under stringent conditions to a secondnucleic acid from an isolated human herpes virus having the morphologyof a human herpes virus and a double-stranded DNA genome of about 170Kb, wherein genomic DNA from said isolated human herpes virus hybridizesunder stringent conditions with nucleic acid of molecular clone ZVH14(ATCC Accession No. 40,247); and further wherein said first nucleic aciddoes not hybridize under said stringent conditions with the nucleic acidof:(a) Epstein-Barr virus; (b) human cytomegalovirus (CMV); (c) HerpesSimplex virus (HSV); (d) Varicella-Zoster virus (VZV); or (e) Herpesvirus saimiri.
 2. The polypeptide of claim 1, wherein said polypeptideis specifically bound by antibodies in human serum from a patientinfected with the isolated human herpes virus.
 3. A compositioncomprising antigenic protein of an isolated human herpes virus havingthe morphology of a human herpes virus and a double-stranded DNA genomeof about 170 Kb, wherein genomic DNA from said isolated human herpesvirus hybridizes under stringent conditions with nucleic acid ofmolecular clone ZVH14 (ATCC Accession No. 40,247); and further whereinsaid first nucleic acid does not hybridize under said stringentconditions with the nucleic acid of:(a) Epstein-Barr virus; (b) humancytomegalovirus (CMV); (c) Herpes Simplex virus (HSV); (d)Varicella-Zoster virus (VZV); or (e) Herpes virus saimiri.